Spinnen et al. J Nanobiotechnol (2021) 19:83 https://doi.org/10.1186/s12951-021-00830-7 Journal of Nanobiotechnology

RESEARCH Open Access Therapies with CCL25 require controlled release via microparticles to avoid strong infammatory reactions J. Spinnen1* , K. Fröhlich1^, N. Sinner1, M. Stolk1, J. Ringe1, L. Shopperly1, M. Sittinger1, T. Dehne1 and M. Seifert2,3

Abstract Background: Chemokine therapy with C–C motif chemokine ligand 25 (CCL25) is currently under investigation as a promising approach to treat articular cartilage degeneration. We developed a delayed release mechanism based on Poly (lactic-co-glycolic acid) (PLGA) microparticle encapsulation for intraarticular injections to ensure prolonged release of therapeutic dosages. However, CCL25 plays an important role in immune cell regulation and infammatory processes like T-cell homing and chronic tissue infammation. Therefore, the potential of CCL25 to activate immune cells must be assessed more thoroughly before further translation into clinical practice. The aim of this study was to evaluate the reaction of diferent immune cell subsets upon stimulation with diferent dosages of CCL25 in compari- son to CCL25 released from PLGA particles. Results: Immune cell subsets were treated for up to 5 days with CCL25 and subsequently analyzed regarding their cytokine secretion, surface marker expression, polarization, and migratory behavior. The CCL25 receptor C–C chemokine receptor type 9 (CCR9) was expressed to a diferent extent on all immune cell subsets. Direct stimula- tion of peripheral blood mononuclear cells (PBMCs) with high dosages of CCL25 resulted in strong increases in the secretion of monocyte chemoattractant protein-1 (MCP-1), interleukin-8 (IL-8), interleukin-1β (IL-1β), tumor-necrosis- factor-α (TNF-α) and interferon-γ (IFN-γ), upregulation of human leukocyte antigen-DR (HLA-DR) on monocytes and ­CD4+ T-cells, as well as immune cell migration along a CCL25 gradient. Immune cell stimulation with the supernatants from CCL25 loaded PLGA microparticles caused moderate increases in MCP-1, IL-8, and IL-1β levels, but no changes in surface marker expression or migration. Both CCL25-loaded and unloaded PLGA microparticles induced an increase in IL-8 and MCP-1 release in PBMCs and macrophages, and a slight shift of the surface marker profle towards the direc- tion of M2-macrophage polarization. Conclusions: While supernatants of CCL25 loaded PLGA microparticles did not provoke strong infammatory reac- tions, direct stimulation with CCL25 shows the critical potential to induce global infammatory activation of human leukocytes at certain concentrations. These fndings underline the importance of a safe and reliable release system

*Correspondence: [email protected] 1 Tissue Engineering Laboratory, BIH Center for Regenerative Therapies, Department for Rheumatology and Clinical Immunology & Berlin Institute of Health at Charité–Universitätsmedizin Berli, BCRT​, Charitéplatz 1, 10117 Berlin, Germany Full list of author information is available at the end of the article K. Fröhlich—deceased

© The Author(s) 2021. This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creat​ iveco​ ​ mmons.org/​ licen​ ses/​ by/4.​ 0/​ . The Creative Commons Public Domain Dedication waiver (http://creat​ iveco​ mmons.​ org/​ publi​ cdoma​ in/​ ​ zero/1.0/​ ) applies to the data made available in this article, unless otherwise stated in a credit line to the data. Spinnen et al. J Nanobiotechnol (2021) 19:83 Page 2 of 16

in a therapeutic setup. Failure of the delivery system could result in strong local and systemic infammatory reactions

that could potentially negate the benefts of chemokine therapy.

Introduction proliferation-promoting secretome [3–5]. However, the Research in the feld of regenerative medicine is increas- efcacy of these treatments remains very limited and the ingly revealing the enormous potential of the human results may not justify the cost of an ex vivo cell ther- body’s inherent self-healing powers. Chronic tissue dam- apy. To enhance the cost-efectiveness and efcacy of age, as seen in conditions such as osteoarthritis (OA) are MSC targeting therapies, the application of chemokines not ignored by the body but mechanisms to repair dam- embedded in certain release devices are of growing age are constantly being attempted. Unfortunately, these interest. Chemokine enriched biopolymers enable both repair processes are often not perfectly synchronized mechanical closure of the lesion as well as delayed release with the underlying pathological conditions or are nega- of their regenerative factors [6, 7]; an approach called in- tively afected by the infammatory processes present. In situ tissue engineering [8]. Certain chemokines have been OA, for example, chondrocytes in the deeper cartilage shown to both attract certain cells like MSCs to the site tissue respond to degeneration of the outer tissue layers of application, as well as increase the tissue producing with increased expression of cartilage-specifc matrix capacities of the resident cells. So far, multiple chemokine proteins such as collagen II and aggrecan. However, these candidates are under investigation for their suitability, mechanisms are not sufcient to repair the tissue damage with C–X–C motif chemokine ligand 12 (CXCL12) and [1, 2]. IL-8 being among the most popular [9, 10]. Terefore, the tissue requires a general change in In previous work, we identifed C–C motif chemokine microenvironment to modulate chronic infammation ligand 25 (CCL25) as a potent candidate for therapeutic and provide pro-regenerative stimuli. Besides the injec- chemoattraction of MSCs through broad-based migra- tion of growth factors or instrument-based stimula- tion assays [11, 12]. Because the knee joint is thought tion, multiple studies have shown that the intraarticular to contain many loculated MSCs, and the knee as a pre- application of mesenchymal stromal cells (MSCs) can formed body cavity is well suited for injection therapy, sway the microenvironment towards a pro-regenera- the efcacy of CCL25 for osteoarthritis was investi- tive direction owing to their immune-regulatory and gated. In an in vivo OA model (Dunkin-Hartley guinea Spinnen et al. J Nanobiotechnol (2021) 19:83 Page 3 of 16

pig), one injection per week of CCL25 dissolved in hya- of PLGA is well established [26], there is currently no luronic acid (HA) into afected joints for four consecu- approved treatment that uses free-foating PLGA parti- tive weeks resulted in up to 28% less cartilage damage cles in human joints. It is also conceivable that the bio- (graded by the histopathological OARSI Score) in the material itself could cause undesired infammatory side CCL25-treated joint compared with the untreated joint efects related to its acidic properties. and HA and sodium chloride (NaCl) control groups To address these issues, we conducted an immunologi- [13, 14]. After proof of concept, a release system based cal study assessing the interaction of PLGA/CCL25 with on poly (lactic-co-glycolic acid) (PLGA) microparticles diferent human immune cell subsets. Te objective of was developed that allows a single injection followed by this study was to assess the infammatory potential of controlled, sustained release of CCL25 into the joint over PLGA/CCL25 to induce typical changes that leukocytes several weeks. Importantly, microparticles with diam- show during an infammatory response. In our analysis, eters of ~ 50–100 µm allow for intraarticular injection we focused on the potential alteration of pro-infamma- using a syringe instead of arthroscopic implantation. On tory cytokine secretion, the regulation of cellular activa- the one hand, this saves the patient a hypothetical inva- tion and polarization markers, and leukocyte migration sive procedure, on the other hand, the PLGA degradation induced by CCL25 or PLGA. kinetics enable steady level of chemokines to recruit suf- fcient amounts of cells while avoiding potentially harm- Results ful peak concentrations that could result from multiple Immune cell subsets exhibit difering expression levels injections [15, 16]. PLGA was chosen because it is already of CCR9 successfully used as a microencapsulation material (also Te determination of CCR9 expression is an important in the form of microparticles [17, 18]), encapsulation of prerequisite for enabling CCL25-induced cell migra- proteins is well standardized and due to its biocompat- tion and intracellular signaling as it is the receptor for ibility and biodegradability is considered safe for use the ligand CCL25. Tus, extracellular CCR9 expres- by international regulatory authorities—demonstrated sion was investigated using fow cytometry on difer- by numerous approvals of PLGA-based drugs [19–21]. ent immune cell subsets by staining with a fuorescently PLGA is degrading delayed due to the hydrolysis of its labeled antibody (Fig. 1a). Peripheral blood mononu- ester linkages in presence of water. Te resulting carbox- clear cells (PBMCs), monocytes and ­CD4+ T-cells exhib- ylic acid chain ends of the cleavage further may further ited a mean CCR9 expression of 31 ± 6%, 45 ± 2% and + accelerate the process and lead to autocatalysis [22]. As a 34 ± 3%, respectively. ­CD8 T-cells showed the lowest result of internal difusion, a pH gradient from the center mean surface expression, with 21 ± 4%. Te strongest to the surface of the particle is forming and degradation CCR9-expression was found on M1-type macrophages products and will difuse and erode away from the entire with around 78%. Unpolarized M0-type macrophages volume of the particle (bulk erosion). As PLGA degra- showed a seemingly lower CCR9 expression of about 34% dation is driven by hydrolysis and degradation products (Fig. 1a). (glycolic and lactic acid) are non-toxic chemicals that can be metabolized by the body, it can be used as delivery Stimulation with high dosages of CCL25 induces high system in physiological environments [23]. Furthermore, secretion levels of pro‑infammatory cytokines in PBMCs PLGA is commonly used in the knee cavity for autolo- To assess the unmediated reaction of diferent leuko- gous chondrocyte transplantation, suture material and cyte subsets to CCL25, PBMCs and diferently polarized not known to interact with the joint tissue in any harmful macrophages were stimulated over fve days with dos- way. Since the expiration of frst patents on PLGA micro- ages of 10, 100 and 750 nM CCL25 (Fig. 1b, c). Superna- particulate systems, the development of generic prod- tants were collected on day one, three, and fve, and the ucts experiencing a revived interest in pharmaceutical cytokines present were analyzed with the LEGENDplex research [24]. immunoassay. When testing diferent macrophage sub- Advances in chemokine therapy are hampered by sets, unpolarized M0 macrophages reacted strongly and insufcient clinical biosafety data. Analysis of existing dose-dependently to CCL25 stimulation with an up to experimental data on CCL25 revealed strong involve- tenfold increase in IL-8 secretion in the 750 nM group ment in adaptive immune system development, dys- (p = 0.08), while M1-polarized macrophages showed a functional immunological processes in endometriosis, small increase independent of the dose used (Fig. 1b). infammatory bowel disease, and rheumatoid arthritis Direct stimulation of PBMCs with CCL25 leads to a very [25]. Hence, application of this chemokine could cause a strong and dose-dependent secretion of the pro-infam- signifcant infammatory response and potentially cause matory chemokines and cytokines IL-8, MCP-1, TNF- more harm than good. Also, while the biocompatibility α, IL-1β, interleukin-6 (IL-6) and IFN-γ (Fig. 1c). While Spinnen et al. J Nanobiotechnol (2021) 19:83 Page 4 of 16

Fig. 1 CCR9 expression and secretion profle of immune cell subsets after stimulation with CCL25. PBMCs, unpolarized (M0) and polarized macrophages (M1) were stained with a CCR9-specifc antibody and analyzed by fow cytometry. PBMCs and macrophages were stimulated with diferent dosages of CCL25 or left unstimulated as a negative control. A LEGENDplex cytokine array was performed using the culture supernatants after day 1, day 3 and day 5. a The expression of the CCL25-receptor CCR9 on diferent immune cell subsets is shown as mean of the percentage of marker positive cells SD (n 1–4). b Shown are the cytokine/chemokine release data for M0 and M1 macrophages after one-day stimulation with + = 10, 100 and 750 nM CCL25 as the mean fold change SD compared to the negative control (red dotted line) (n 4). c The cytokine/chemokine + = release data for PBMCs stimulated with 10, 100 and 750 nM CCL25 over three days are displayed as the mean fold change SD normalized to + the negative control (red dotted line) (n 3). Signifcance was analyzed using either a student’s T-test or the Mann–Whitney-U test depending = on whether data followed normal distribution. Individual p-values are given above the bars if lower than p 0.20. PBMCs:peripheral blood = mononuclear cells, ΜΦ Macrophages

for most cytokines/chemokines there were only moder- cytokines measured on the ffth day showed lower values ate increases in the 10 nM and 100 nM CCL25 stimula- than on day 1 after stimulation. tion groups, PBMCs reacted with a strong increase in all tested mediators for the 750 nM stimulation group. CCL25 causes a dose‑dependent upregulation While IL-8 also showed a statistically signifcant increase of the activation marker human leukocyte antigen‑DR in secretion after being triggered with lower CCL25 con- (HLA‑DR) on immune cells centrations, the other cytokines increased exclusively Flow cytometry was used to investigate the efects of after stimulation with the highest CCL25 concentra- CCL25 on the activation and polarization of immune tion. On day 5 of stimulation, secretion of cytokines and cells. Macrophages were frst analyzed for the diferential chemokines seemed to decline. Apart from IFN-γ, the expression of characteristic polarization markers for the Spinnen et al. J Nanobiotechnol (2021) 19:83 Page 5 of 16

M1 (CD80 and HLA-DR) or M2 direction (CD163 and with a signifcant HLA-DR upregulation to CCL25 stim- CD206). Analysis of the activation marker HLA-DR was ulation. Cytotoxic ­CD8+ T-cells showed weaker changes then performed on PBMCs. While no statistically signif- in the expression level of HLA-DR than CD4­ + cells and cant changes in the expression of either M1 or M2 type did not reach a statistically signifcant diference com- characteristic polarization markers on macrophages were pared to the unstimulated negative control. detected (data not shown), PBMCs showed a very clear HLA-DR upregulation when exposed to all three concen- Macrophages and T‑cells show dose dependent migration trations of CCL25 (Fig. 2a). ­CD45+CD3− negative cells towards CCL25 (all mononuclear leukocytes besides T-cells) exhibited Assessing CCL25′s capabilities to recruit leukocytes from the strongest reaction, with a dose-dependent increase foreign tissues, diferent macrophage and T-cell subsets up to a 1.8-fold change compared to the negative control. were examined in a Boyden-Chamber migration assay Also, ­CD4+ cells reacted in a dose-dependent fashion employing a CCL25 concentration range from 0.01 to

Fig. 2 Surface marker changes and migration behavior of immune cell subsets after CCL25 stimulation. After stimulation with diferent dosages of CCL25 (10, 100, 750 nM), immunofuorescence staining with antibody panels and subsequent fow cytometry was performed to detect leukocyte activation and macrophage polarization based on surface marker expression. Additionally, a Boyden-Chamber migration assay was performed with a CCL25 dilution series (0.01–1000 nM). a HLA-DR expression levels of diferent immune cell subsets stimulated with 10, 100 and 750 nM CCL25 over three days are shown as the mean of the percentage of marker positive cells SD normalized to the negative control (red dotted line); + n 3. b The migration of immune cell subsets towards a dilution series of CCL25 is displayed as the mean SD of the number of migrated cells = + (n 4). Macrophage polarization through CCL25 stimulation was analyzed by immunofuorescence antibody staining and fow cytometric analysis = of M0 and M1 macrophages against either c M1 polarization markers CD80 and HLA-DR or d M2 polarization markers CD163 and CD206 (n 4). = Surface marker expression levels are shown as the mean SD of normalized values compared to the negative control (red dotted line). Statistical + signifcance was analyzed using either a student’s T-test or the Mann–Whitney-U test depending on whether data followed a normal distribution. Individual p values are given above the bars if lower than p 0.20. ΜΦ Macrophages = Spinnen et al. J Nanobiotechnol (2021) 19:83 Page 6 of 16

1000 nM (Fig. 2b). After CCL25 stimulation, both unpo- with IL-8, MCP-1 secretion on day 5 equalized to similar larized M0- and M1-polarized macrophages reacted with levels at about 1.5-fold of the negative control, but only a dose-dependent increase in migration. While migration the 0.5% w/v concentration of the CL particle reached towards CCL25 in unpolarized M0-macrophages already statistical signifcance (1.7 ± 0.5-fold change; p = 0.02). started at concentrations of 500 nM, M1 macrophages Macrophages also responded to CCL25 stimulation by only showed signifcant migration towards the highest increasing secretion of IL-8 but did not increase secre- tested concentration of 1000 nM. T-cells also showed a tion of MCP-1 or other cytokines (Fig. 3b). M1-polar- strong increase in migration towards CCL25 concentra- ized macrophages reacted similarly to 0.5% and 0.05% tions of 500–1000 nM. Te migration of CD8­ + T-cells w/v of either CL or NL particles, with a slight increase + appeared to be stronger than that of ­CD4 T-cells. in IL-8 secretion (CL: 0.5% w/v: 1.49 ± 0.68; 0.05% w/v: 1.48 ± 0.76; NL: 0.5% w/v: 1.83 ± 0.72; 0.05% w/v: Macrophages do not polarize or change their polarization 1.55 ± 0.35). Unpolarized M0 macrophages reacted with upon CCL25 stimulation a much stronger overall secretion of IL-8 and an observa- M0- and M1-polarized macrophages were stimulated ble diference between CCL25-loaded and unloaded par- with 10, 100 and 750 nM CCL25, and then analyzed for ticles (CL: 0.5% w/v: 4.42 ± 3.4; 0.05% w/v: 3.37 ± 4.0; NL: their expression of surface markers specifc for M1 or M2 0.5% w/v: 2.73 ± 3.2; 0.05% w/v: 3.11 ± 3.0). macrophage polarization via fow cytometry (Fig. 2c). M0 macrophages showed no change in CD80 and HLA-DR Both CCL25‑loaded and unloaded particles modulate (M1 markers), no signifcant change in CD206 and a sig- the M1 macrophage phenotype nifcant decrease in CD163 (M2 marker) expression. In Next, we determined whether the unloaded or CCL25- summary, no clear trend in the polarization mediated by loaded particles could infuence the polarization status of CCL25 could be identifed. human macrophages in vitro and even reverse the devel- opment of a pro-infammatory M1 phenotype. Both the Immune cell stimulation by CCL25‑loaded and unloaded CL and NL particle groups caused a signifcant reduc- particles leads to increased IL‑8 and MCP‑1 secretion tion of CD80 expression, a typical M1 marker, in cultures Similar to the previous CCL25 stimulation assay, human of M1-polarized macrophages by a mean of 1.52 ± 0.07 leukocytes were incubated with undegraded CCL25- (Fig. 3c). However, the second M1 marker assayed, HLA- loaded (CL) and unloaded (NL) PLGA particles in two DR, showed no signifcant changes in expression level. At diferent concentrations (0.5% w/v and 0.05% w/v) to the same time, expression of the characteristic M2 sur- distinguish between the efect of CCL25 and intact par- face marker CD206 in co-cultures of already polarized ticle, as well as to check the integrity of the loaded parti- M1 macrophages with the particles trended towards an cles. Cytokine levels of PBMC cultures treated with both increase with a fold change of 1.50 ± 1.57. Expression of particle types increased for IL-8 and MCP-1 compared the M2 Marker CD163 trended towards a decrease of to the untreated negative control, peaking around the around 1.20 in both groups with the 0.5% w/v particles third day of stimulation (Fig. 3a). Te strongest reaction reaching p-values of 0.07 and 0.12, respectively (Fig. 3d). for IL-8, albeit not statistically signifcant, was shown by Unpolarized M0 macrophages also showed a slight the 0.5% w/v NL particles on the third day with a mean reduction of CD80 and CD163 after particle incubation, but no increase in CD206 (Fig. 3b, d). No signifcant dif- increase of 6.48 ± 6.38-fold change in secretion (p = 0.14). In relation to the negative control, the mean increase in ferences between the loaded and unloaded particles were IL-8 for 0.5% w/v CL particles was, at 4.2, much lower observed. than for the NL particles. Stimulation with 0.05% w/v of either CL or NL particles induced very similar fold Immune cell stimulation with supernatants changes for IL-8 secretion with the CL particles causing of CCL25‑loaded particles causes an increase in IL‑8, MCP‑1 a slightly higher IL-8 secretion on the third day than the and IL‑1β secretion NL particles (CL = 5.2 ± 3.98; NL = 4.26 ± 4.84). Inter- To distinguish between the efects of particle fragments estingly, all stimulation groups reached a very similar and the enclosed CCL25 on the leukocytes, CL and NL level of IL-8 secretion on the ffth day, with around four particles were both allowed to degrade over either 21 times that of the negative control. For MCP-1, all groups or 63 days. Te supernatants of degraded particles were except the 0.05% w/v concentration of the NL particles applied to the PBMCs in functional in vitro assays, and the showed high increases in secretion on the third day. Te cytokine secretion profle was determined at day 3 and 5 highest mean increase was seen in 0.05% w/v CL parti- after supernatant stimulation (Fig. 4a). PBMC cultures cles (4.5 ± 3.3-fold change; p = 0.22) followed by the 0.5% treated with particle supernatants showed higher secretion w/v NL particles (4.3 ± 3.3-fold change; p = 0.35). As seen levels of IL-8, MCP-1 and IL-1β compared to the control. Spinnen et al. J Nanobiotechnol (2021) 19:83 Page 7 of 16

Fig. 3 Response profle of immune cell subsets after stimulation with CCL25-loaded/unloaded PLGA particles. PBMCs and macrophages were stimulated with CCL25-loaded (CL) and unloaded (NL) PLGA particles at two diferent concentrations (0.5% w/v and 0.05% w/v). Induced changes in the cytokine/chemokine secretion profles of PBMCs and macrophages were analyzed with a LEGENDplex cytokine array. In addition, unpolarized M0 macrophages and polarized M1 macrophages were stained for characteristic surface markers with fuorescently labeled antibodies and measured by fow cytometry to analyze their polarization status. a Cytokine release data (IL-8, MCP-1, IL-1) of PBMCs stimulated with 0.5% w/v and 0.05% w/v of CL and NL PLGA particles over 5 days are shown as mean fold change SD normalized to the negative control (red dotted line) + (n 3). b Cytokine release data (IL-8, MCP-1, IL-1) of M0/M1 macrophages stimulated with 0.5% w/v and 0.05% w/v of CL and NL PLGA particles over = 1 day are shown as the mean fold change SD normalized to the negative control (red dotted line) (n 4). Surface expression levels of M0 (left) and + = M1 (right) macrophages for the characteristic M1 polarization markers CD80 and HLA-DR (c) and for M2 polarization markers CD206 and CD163 (d) after stimulation with 0.5% w/v and 0.05% w/v of CL and NL PLGA particles over 1 day are displayed as mean of the fold change SD normalized + to the control (red dotted line), (n 4). Statistical analysis was performed using either a student’s T-test or the Mann–Whitney-U test depending = on whether data showed normal distribution. Individual p-values are given above the bars if they are lower than p 0.20. CL CCL25-loaded, NL = non-loaded, PC Particle concentration., w/v weight per volume, ΜΦ Macrophages

In the CL group, IL-8 secretion was increased 4.6-fold (CL: 21d: 1.75 ± 1.85; 63d: 1.81 ± 1.68; NL: 21d: 1.54 ± 1.47; ( 5.3, p 0.22) after treatment with the 21-day superna- ± = 63d: 1.6 ± 1.58). However, after fve days, secretion of IL-1β tant and 5.3-fold (± 7.3, p = 0.45) with the 63-day superna- in PBMCs stimulated with the 21-day CL supernatant tant. Te latter NL supernatants only induced a 5.14-fold increased by a statistically signifcant amount (1.77 ± 0.27: ( 7.4, p 0.49) increase in IL-8 levels, which does not ± = p = 0.02), while for the corresponding NL particle superna- reach statistical signifcance. IL-1β secretion increased in tants, the IL-1β secretion tended to drop down to the levels a statistically insignifcant manner by roughly the same of the negative control (0.95 ± 0.46). Te same dynamics amount in the CL and NL particle groups after three days were observed for MCP-1 secretion, with a strong increase Spinnen et al. J Nanobiotechnol (2021) 19:83 Page 8 of 16

(See fgure on next page.) Fig. 4 Reaction of immune cell subsets after stimulation with supernatant from degraded CCL25-loaded/unloaded PLGA particles. CCL25-loaded (CL) and unloaded (NL) PLGA particles were allowed to degrade for either 21 or 63 days and particle supernatant was collected and used for in vitro stimulation assays with human PBMCs and macrophages. Changes in the cytokine/chemokine release pattern of PBMCs and macrophages were analyzed with a LEGENDplex cytokine array. Additionally, macrophages were analyzed by immunofuorescence staining and fow cytometry for the expression of surface markers indicating activation or polarization. a Cytokine array of PBMCs stimulated over 5 days with 21- and 63-day CL and NL PLGA particle supernatants. Cytokines with detectable diferences in fold change are IL-8, MCP-1 and IL-1β. Data is shown as the mean fold change SD normalized to the negative control (red dotted line) (n 3). b Cytokine array of M0/M1 macrophages stimulated over 1 day with + = 21- and 63-day CL and NL PLGA particle supernatants. Cytokines with detectable diferences in fold change are IL-8 and MCP-1. Data is shown as mean fold change SD normalized to the negative control (red dotted line) (n 4). c The migration of immune cell subsets towards a dilution + = series of particle degradation supernatant is displayed as the mean SD of the number of migrated cells (n 4). d/e Flow cytometric analysis of M0/ + = M1 macrophage polarization surface markers after 1 day of stimulation with 21- and 63-day CL and NL PLGA particle supernatants. Increased CD80 and HLA-DR indicates M1 polarization (d), while increased CD163 and CD206 indicates M2 polarization (e). Results are displayed as mean of the fold change SD normalized to the control (red dotted line), (n 4). Statistical analysis was performed using either a student’s T-test or the Mann– + = Whitney-U test depending on whether data followed normal distribution. Individual p-values are given above the bars if lower than p 0.20. CL = CCL25-loaded, NL non-loaded, NC negative control, ΜΦ Macrophage after three days in all groups, and with high levels main- characteristic markers were analyzed by fow cytome- tained on the ffth day in the CL particle supernatant try. After treatment of M1-polarized macrophages with group. At the same time, MCP-1 secretion decreased in CL and NL particle supernatants, HLA-DR expression the NL particle supernatant group (Fig. 4a). Again, mac- was signifcantly increased with a mean fold change of rophages were also analyzed in the same way, but only 1.4 ± 0.2 (Fig. 4d). However, there was no observable dif- showed higher secretion levels for IL-8. Te 21-day and ference between the groups treated with CCL25-loaded- 63-day old CL particle supernatants caused a 1.4-fold and unloaded particle supernatant and the timespan of (± 0.5, p = 0.32)/1.43 (± 0.83, p = 0.44) increase in cultures degradation. Meanwhile, the M1 marker CD80 decreased of M0 macrophages and a 1.15 (± 0.29, p = 0.43) / 1.29 by a mean fold change of 1.3 (± 0.17). M0-polarized mac- (± 0.04, p = 0.03) increase in M1 macrophages, respectively rophages also increased expression of HLA-DR after (Fig. 4b). Interestingly, IL-1β secretion from M1-polarized supernatant stimulation from either CL or NL parti- macrophages was signifcantly lower in all CL and NL cles, but did not reach statistical signifcance. However, supernatant groups. changes in the expression level of the M2 markers CD206 and CD163 induced by the particle supernatant treat- Supernatants of both loaded and unloaded particles cause ment were not observable in either M0 or M1 pheno- no signifcant increases in immune cell migration types (Fig. 4d). To assess the chemoattractive potential of the particle com- ponents, the supernatants of degraded particles (21 and Discussion and limitations 63 days) were used as stimuli in a Boyden-Chamber migra- Te objective of this study was to analyze the infam- tion assay with the diferent leukocyte subsets (Fig. 4c). matory potential of a novel therapy for OA based on Stimulation was performed in a 1000-factor dilution series CCL25-loaded PLGA particles. We wanted to examine to distinguish between the efects of CCL25 and PLGA the reaction of leukocytes involved in innate and adaptive fragments. When testing CD4­ + T-cells, undiluted super- immune responses towards each individual component natants of both particle groups reduced the migration ten- of the potential therapeutic agent to better assess which fold, which is a statistically signifcant decrease. Also, M1 subcomponents are potentially critical and at what doses. macrophages showed a statistically signifcant reduction in For this purpose, human PBMCs isolated from whole migration at 1:10 and 1:100 dilutions of the particle super- blood and macrophages generated in vitro were exposed natants, but only in the NL group. Te other tested immune to diferent dosages of (i) pure CCL25, (ii) CCL25-loaded cell subsets (M0 macrophages, ­CD8+ T-cells) showed no PLGA particles and (iii) their supernatants after vary- signifcant diferences in migration in response to the parti- ing time points of particle degradation. We subsequently cle supernatants compared to the control (Fig. 4c). analyzed the changes in pro-infammatory cytokine secretion, the leukocyte activation and macrophage Supernatants of both loaded and unloaded particles polarization by monitoring characteristic cell surface increase HLA‑DR expression on macrophages markers and investigated immune cell migration. Our To assess the capacity of the particle supernatants to results provided new insights regarding the infammatory modulate the activation or polarization state of human potential of a possible PLGA-based delivery chemokine macrophages, the surface expression levels of the therapy for the treatment of OA. Crucially, the CCL25 Spinnen et al. J Nanobiotechnol (2021) 19:83 Page 9 of 16

receptor CCR9 was found to be expressed on all analyzed TNF-α, IL-1β, IL-6 and IFN-γ from PBMCs. Tis com- leukocyte subsets, which makes a reaction upon contact bination of cytokines indicates a general, uncontrolled with this chemokine very likely and underlines the need leukocytic activation. Except for IFN-γ, secretion levels for a thorough analysis of this interaction. of all cytokines start to drop down after 5 days of stim- Te dose-dependent reaction of diferent leukocyte ulation. However, these cytokines were considered to subsets to stimulation with CCL25 was remarkably clear. be among the main biological drivers in the pathogen- While the immune cells reacted only slightly to CCL25 esis of OA. Especially MCP-1 and IL-8 are reported to concentrations of 10 and 100 nM, stimulation with correlate with clinical levels of pain and knee swelling. 750 nM CCL25 caused a strong secretion of MCP1, IL-8, Concerningly, these two cytokines exhibited the largest Spinnen et al. J Nanobiotechnol (2021) 19:83 Page 10 of 16

increase in expression for all dosages of CCL25 in both on the frst day of stimulation with loaded particles. PBMCs and macrophages [27–29]. In an in vivo situa- Overall, however, the diference between the loaded and tion, this high level might subsequently cause an increase unloaded particles was minute. It is very likely, that the in infammation by damaging the surrounding tissue and reaction was mostly caused by the PLGA, with a mar- possibly further recruiting more immune cells to the site. ginal CCL25 contribution. Te observed increase in IL-8 Flow cytometric analysis of HLA-DR expression revealed secretion could be induced by the acidic degradation a high sensitivity, especially for monocytes and ­CD4+ of the PLGA, which has been shown to cause IL-8 pro- T-cells, to CCL25 stimulation, with visible increases in duction in other cells [32, 33]. Te infuence of CCL25- expression even at low concentrations of 10 nM CCL25. loaded particles on the polarization of macrophages did Tis most likely contributes to the strong cytokine secre- not show a very clear polarization trend towards either a tion in PBMCs through the simultaneous activation of more pro-infammatory M1-type or the pro-regenerative both monocytes and ­CD4+ T-cells, resulting in an ampli- M2-type. However, the decrease of CD80 and increase of fcation efect. Tis would also explain why macrophages CD206 point in the direction of increased M2 polariza- alone do not respond as strongly as PBMCs, as they were tion. Macrophage polarization towards the pro-regen- cultured in isolation from other cell types and probably erative M2 macrophage direction by PLGA has been not exposed to a strong paracrine co-stimulus. CCL25 recently described by others [34, 35]. M2 macrophage also exhibited the critical potential of recruiting leuko- polarization is thought to be a highly favorable feature cytes, which was expected given the physiologic role of of tissue engineering devices to decrease infammation CCL25 in T-cell homing [30]. Previous studies already and promote healing [36, 37]. Tis in turn underlines demonstrated the capability of injected CCL25 to recruit the suitability of PLGA as a delivery platform for regen- T-cells to the site of injection [31]. Beginning at a certain erative therapeutics. Overall, these results are in line with concentration, unpolarized M0 macrophages and M1 the existing literature. PLGA has been used clinically as polarized macrophages as well as ­CD4+ and CD8­ + T-cells a surgical suture material for years and is not known to started to strongly migrate towards CCL25 concentra- cause major infammatory side efects other than the tion gradients. Te critical dose for migration in vitro aforementioned acidic degradation. On the contrary, was between 500–1000 nM, which roughly corresponds the promotion of M2 macrophage polarization might to the concentrations that also triggered cytokine secre- even prove advantageous to minimize an already existing tion. Te level of HLA-DR expression induced by CCL25 infammation. stimulation correlates with the CCR9 expression profle, After stimulation with supernatants from the degraded with monocytes revealing the highest CCR9 expression CCL25-loaded particles, PBMCs showed a much and HLA-DR upregulation, while CD8­ + T-cells showed stronger reaction induction of cytokine secretion than the lowest CCR9 expression and HLA-DR upregulation. after the stimulation with undegraded but loaded par- It is also likely that the strong HLA-DR upregulation ticles. Considering the release pattern exhibited by the observed is due to co-stimulation by secreted cytokines. particles (Fig. 5c), supernatants at day 21 and 63 contain Since the macrophages were cultivated as monocultures, roughly 75% and 100% of released chemokine, respec- they could not acquire a co-stimulus from other leuko- tively. Tis amounts to a maximum of 352 nM (63 days) cytes. Tis might explain that they show no HLA-DR 35.2 nM (21 days) of CCL25. Te supernatants contained upregulation, despite carrying high amounts of CCR9 both the degraded PLGA fragments and the chemokine on their surface. In summary, CCL25, starting at concen- CCL25, and triggered release of IL-8 and MCP-1 by trations of 250–500 nM, has a strong potential to trig- immune cells as well as IL-1β in addition to the parti- ger infammatory processes by recruiting and activating cles. Notably, the supernatants of CCL25 loaded particles leukocytes and inducing cytokine secretion, especially caused a higher and longer secretion of these cytokines. in a heterogeneous cell environment such as chronically IL-1β is a highly potent pro-infammatory cytokine, of infamed tissue or blood. which only a few nanograms are sufcient to cause mas- When analyzing the immune cell responses to the sive local infammation and tissue degradation, especially CCL25-loaded PLGA particles, the induced response in human joint tissue [38–41]. Tese fndings must be was much weaker compared to the direct CCL25 stimu- considered in the context of the in vivo situation. CCL25 lation. Te CCL25-loaded particles themselves triggered as well as PLGA would also be degraded enzymatically the release of cytokines such as IL-8, MCP-1 and IL-1β over time, meaning that particle supernatants generated in PBMCs, and IL-8 in the M0 and M1 macrophage in vitro contained much more CCL25 and undegraded subsets. Te increase of IL-8 and MCP-1 was slightly fragments than would occur in a therapeutic situation. stronger and achieved more statistical signifcance in Further, compared to the direct stimulation with CCL25, the CCL25-loaded particle group. IL-1β only increased the supernatants of the degraded particles did not cause Spinnen et al. J Nanobiotechnol (2021) 19:83 Page 11 of 16

Fig. 5 Characteristic features of PLGA particles. PLGA particles were analyzed morphologically with SEM, evaluated for endotoxin contamination with the Limulus amebocyte lysate (LAL) test, and functionally assessed by their CCL25 release profle. Representative SEM images of the PLGA particles generated are shown in a i,ii with 40 and 200 magnifcation at day one, iii, iv with 40/ 200 magnifcation after 14 days of × × × degradation, v, vi with 40/ 200 magnifcation after 28 days of degradation and vii, viii with 1000 magnifcation after 63 days of degradation. × × × Scale bars represent 500 and 100 µm, respectively. b The results of the LAL test for the detection of endotoxins are shown for diferent stock solutions of CCL25 (10 and 500 nM) and the supernatant from PLGA particles (CL supernatant) compared to the test standards (0.1–10 nM). c The cumulative release in percent SD of CCL25 from PLGA particles over time until day 63 (n 3) was determined by ELISA. CL CCL25-loaded, PLGA ± = Poly (lactic-co-glycolic acid)

an increase in HLA-DR expression on immune cells or that were observed in vitro did not difer much from the enhance their migration. Tis does suggest that, even unloaded particles, it can be expected that particles care- though the theoretical concentration of CCL25 in the fully loaded with CCL25 are most likely safe to use. In supernatants amounted up to 352 nM, due to the protein fact, the secretion of IL-8 and MCP-1 after stimulation degradation (average half-time of proteins in aqueous of leukocytes by polylactic scafolds has been previously phases is about 5–7 days) [42] the proportion of active described by Caires et al. [43] as potentially benefcial CCL25 always remains below the presumed critical con- for healing by aiding in MSC recruitment. Nevertheless, centration of 100 nM where defnitive infammatory acti- it must be assured that the release device used is tear vation of immune cells becomes visible. However, the resistant. A sudden release of large amounts of CCL25 concentration of CCL25 previously applied in vivo and could prove highly counterproductive in the therapy identifed as benefcial was approximately 1–5 nM [13]. of OA, as it increases rather than reduces intraarticular Concerning cytokine release and leukocyte chemoat- infammation. traction, the frst concentrations which prove to be criti- Te strength of this study is the broad spectrum of dif- cal lie slightly above 100 nM. Our fndings suggest that ferent analytic methods and leukocyte subsets employed, the intra-articular concentration should never exceed which made it possible to draw a diferentiated picture these values, which can be assured by controlled release of the infammatory potential of the diferent compo- from an implant with a slow release kinetic. Since the nents of a CCL25-loaded particle for potential OA treat- leukocyte responses to CCL25-loaded PLGA particles ment. Te study is limited by the relatively low number Spinnen et al. J Nanobiotechnol (2021) 19:83 Page 12 of 16

of human immune cell donors and the strong variability 7 W. For fabrication of big-sized microparticle formula- which impeded the generation of statistically valid state- tions, the primary emulsion was subsequently emulsifed ments due to high standard deviations. Nevertheless, the into 80 ml of the external aqueous w2 phase (5% (w/v) analysis of diferent leukocyte subgroups as well as dif- poly(vinylalcohol) (PVA,MW 67,000, Sigma-Aldrich)). ferently polarized macrophages allowed detailed insights In order to yield small-sized microparticles of formula- into the type and extent of the expected infammatory tion, the primary emulsion was frst emulsifed in 2 ml reaction and sheds light on the complex mechanisms of 5% PVA by vortex mixing at 2500 rpm for 3 min and the signal transduction of the chemokine CCL25 in the con- resulting w1/o/w2 double emulsion was further poured text of a potential clinical application. into 30 ml 1% PVA. Te double emulsions were stirred continuously for 3 h for solvent evaporation. Te solidi- Conclusion fed microparticles were centrifuged and rinsed three Delayed intra-articular release of CCL25 is a promising times with distilled water. In addition to CCL25-loaded approach for a regenerative therapeutic to treat cartilage particles (CL-particles), a batch with non-loaded parti- degeneration in OA. PLGA appears to have only mild cles (NL-particles) was also produced to serve as negative few pro-infammatory properties and may even induce control. After production, the particles were freeze-dried benefcial M2 macrophage polarization. However, high over-night and stored at 4 °C. Te release samples were doses of CCL25 indeed do carry the risk of inducing an generated by incubating the particles at 37 °C in a shaker extensive infammatory response by immune cells pre- at 25 rpm. Sampling was performed after centrifugation sent in joint tissue. We demonstrated for the frst time, for 5 min at 600g by collecting the supernatant, which that externally applied CCL25 is capable to induce secre- was stored at −20 °C. Te samples were transferred into tion of proinfammatory cytokines, including TNF-α, pre-weighed tubes and the collected volume was deter- IL-1β, and IFN-γ in PBMCs, HLA-DR upregulation on mined with a precision scale. It was assumed that 1 g supernatant equaled 1 ml. After collection of the super- monocytes and CD4 + T cells, and enhanced migration natant, fresh medium was added to the particle solution, of CD4 + T-cells and macrophages. Although the previ- ously determined targeted therapeutic doses of CCL25 of and they were placed back in the shaker. Te samples about 1 nM are well below the measured infammatory were collected every seven days beginning with day 7 and cutof of 100 nM—spontaneous disruption of the parti- every 14 days from day 35 to day 63. cles could release a large amount into the joint cavity and produce serious side efects. Due to these potentially dan- gerous side efects, our results suggest very high-quality Morphological analysis of PLGA particles requirements for any CCL25 delivery device. Essentially, To ensure sufcient quality of produced particles, size each batch must be tested for structural integrity in the and surface morphology was analyzed using light micros- frame of tests for spontaneous degradation and resist- copy and scanning electron microscopy (SEM). A small ance to mechanical stress. amount of each PLGA batch was dispersed in Aqua- tex™ (Millipore Sigma, Burlington USA) and 100 to 500 Methods particles were photographed. Light microscopy images ™ Production of CCL25‑loaded PLGA microparticles were captured with the software ProgRes Capture Pro and sampling (Jenoptik, Jena, Germany) and analyzed in ImageJ (Ver- To enclose the hydrophilic chemokine CCL25 (Pepro- sion 1.8.0.112). Te diameters of 10%, 50%, 90% and 99% tech, Rockyhill USA) within the biodegradable polymer ­(DV10, ­DV50, ­DV90, ­DV99) of the particles were obtained. PLGA (Sigma-Aldrich, St. Louis USA), a water-in-oil- ­DV50 of produced particles was at ~ 84 µm, which is within the previously reported range of optimal size in-water ­(w1/o/w2) double emulsion with subsequent solvent evaporation was performed according to a previ- for maximal CCL25 release [15]. Pictures of SEM were ously published method [15]: PLGA polymer (Resomer®, taken with an SEM JFM-6000 electron microscope RG 503—Evonik Industries, Essen, Germany) was dis- (Jeol, Akishima, Japan) at day one and day 63 using the solved (100 mg polymer in 0.7 ml methylene chloride) backscattered electrons (BSE) detector at 200× and and served as organic (o) phase. Te internal aque- 1000× magnifcation. SEM analysis revealed round parti- ous phase (w1) was comprised of 1 µg CCL25 per mg cles, which exhibit few pore formations on an otherwise PLGA that was dissolved in 66.5 ml lactose (300 mM)/ smooth surface. After 63 days, the shape of the previously BSA (0% or 5% w/w)/Tris–HCl (10 mM)-EDTA (1 mM) round particles was completely lost, leaving only frag- bufer. For preparation of the w1/o primary emulsion, the ments or deformed particles, showing the degradation w1-phase was emulsifed in the o-phase either by vortex process is a requirement for functioning CCL25 release mixing at 2500 rpm for 1 min or by sonication for 10 s at (Fig. 5a). Spinnen et al. J Nanobiotechnol (2021) 19:83 Page 13 of 16

Endotoxin detection Streptomycin) containing M-CSF (50 ng/ml Miltenyi Bio- Immune cell reactions are highly inducible by endo- tec, Bergisch Gladbach, Germany), seeded into 6-well cell toxins. To determine if cellular reactions to all tested culture plates, and incubated at 37 °C and 5% CO­ 2. After samples (CCL25, CCL25-loaded PLGA particles and six days of stimulation in culture, monocytes were dif- supernatants of CCL25-loaded PLGA particles) would ferentiated into M0 macrophages. To polarize them fur- be distorted by the presence of endotoxins, an endotoxin ther towards pro-infammatory M1 type macrophages, assay was performed with all stimulation components the cells were detached from the cell culture plates using (10 nM and 500 nM stock solution; supernatants of par- 0.5 ml Accutase (Innovative Cell Technologies Inc., San ticle release) (Fig. 5b). Gram-negative bacterial endo- Diego, USA). Ten, two million cells were seeded in toxin was analyzed by the Pierce™ LAL Chromogenic 6-wells in complete medium supplemented with 20 ng/ Endotoxin Quantitation kit (Termo Fisher Scientifc, ml IFN-γ (Peprotech, Rocky Hill, USA) and 100 ng/ml Waltham, USA) according to manufacturer instructions. lipopolysaccharide (LPS; Sigma-Aldrich, St. Louis–USA) Absorbance at 410 nm was measured on a SpectraMax™ to enable polarization within 48 to 72 h towards the M1 plate reader (Molecular Devices, San Jose, USA). All val- macrophage type. ues remained under 0.1 EU/ml, showing the absence of endotoxin in the samples. Stimulation of immune cell subsets For the analysis of the immune cell secretome, PBMCs CCL25 determination of three diferent donors were seeded in 6-well plates, Te amount of CCL25 released from the PLGA micro- containing two million cells per well in 2 ml of cell com- particles was measured using the human CCL25/TECK plete culture medium. Cells were incubated with the fol- DuoSet® Enzyme linked immunosorbent assay (ELISA) lowing stimuli: (i) diferent concentrations of CCL25 (10, kit (R&D Systems, Minneapolis, USA). Te optical den- 100 and 750 nM), (ii) 10 and 1 mg of CCL25-loaded and sity was measured at 450 nm and 570 nm using the Syn- unloaded particles, and (iii) the supernatants of loaded ergy™ HT Multi-Detection Microplate Reader (BioTek, and unloaded particles that were previously degraded Winooski, USA; Fig. 5c). Te optical imperfections in the over 21 or 63 days (5 mg/ml). Assuming full degrada- plate were corrected for by subtracting the background tion, the maximum achievable concentration in (ii) would at 570 nm from the readings at 450 nm. Te data were amount to 352 and 35.2 nM, respectively. Regarding (iii), evaluated considering the qualitative (blank plus three the theoretical concentration of CCL25 in the superna- times the standard deviation of the blank) and quanti- tants would amount to 75% of total released chemokine tative (blank plus nine times the standard deviation of at 21 days, and 100% at 63 days (Fig. 5c). Tis is also why the blank) borders of the ELISA. CCL25 release started these timepoints were chosen for the supernatants. One slowly on day 14, with more than 90% of total CCL25 well was always left unstimulated to serve as a negative detected between day 21 and day 28. Te strongest control. 200 µl of cell culture supernatants were har- release was observed between days 14 and 21. Recovery vested and stored after 1, 3 and 5 days for further analy- of CCL25 amounted to 77% of incorporated CCL25. sis at − 80 °C. Te same stimulation setting was used for the macrophages, which were only cultivated for 48 h, Isolation and cultivation of human immune cell subsets since expression profles of macrophages already change Peripheral blood mononuclear cells (PBMCs) were iso- after 24 h of culture. For the fow cytometric analysis of lated from bufy coats and human peripheral blood from PBMCs, 0.2 million cells were seeded in 96-well plates healthy volunteers using protocols approved by the eth- containing 200 µl of complete cell culture medium. ics committee of the Charité Universitätsmedizin Berlin (EA2/139/10; EA1/226/14) via density gradient centrifu- Analysis of surface marker expression on immune cells gation using Biocoll (BioTek, Winooski, USA). Lympho- by fow cytometry cytes and monocytes were isolated from PBMCs via Non-adherent PBMCs were resuspended by pipetting, positive selection with antibody-labeled magnetic Micro- and adherent cells were harvested using either a 0.05% Beads (Miltenyi Biotec, Bergisch Gladbach, Germany) trypsin solution with EDTA or Accutase (both Gibco, Life and MACS-columns. 20 μl CD4, CD8 (for the isolation of technology, Termo Fisher Scientifc) and transferred ­CD4+ and CD8­ + T-lymphocytes, respectively) or CD14 to 1 ml Micronic tubes (Micronic). Briefy, cells were (for the isolation of monocytes) MicroBeads per ­107 cells washed once with cold PBS and resuspended in a fnal were added to label the corresponding cell subtypes. volume of 50 µl antibody mix in cold FACS bufer (PBS To generate unpolarized (M0) macrophages, CD14­ + supplemented with 1% FCS) containing the antibody in monocytes were resuspended in complete cell culture the appropriate titrated concentration for 30 min at 4 °C medium (VLE-RPMI, 0.1% AB serum, 1% Penicillin/ in the dark. Incubating cells with only staining bufer Spinnen et al. J Nanobiotechnol (2021) 19:83 Page 14 of 16

Fig. 6 Gating strategies for the surface marker analysis of human peripheral blood mononuclear cells (PBMCs) (a) and macrophages (b). a PBMCs were frst gated (black lined gate) on via sideward scatter area (SSC-A) and forward scatter area (FSC-A) on lymphocytes. Doublets were excluded by gating on forward scatter height (FSC-H) vs. FSC-A and then viable cells were gated by FSC-A vs. Live/Dead Marker. Thereafter, CD3 CD45 T cells + + were identifed based on gating by CD3 vs. CD45, which were then separated into CD4 and CD8 T cells. Finally, the mean fuorescence intensity + + (MFI) of the marker of interest (flled grey histogram) was determined in comparison to the negative control staining (black lined histogram). b Macrophages were frst gated via SSC-A vs. FSC-A to exclude cell debris from the analysis. Doublets were excluded by gating on FSC-H vs. FSC-A. Then, viable cells were identifed (FSC-A vs. Live/Dead) and the MFI of the marker of interest (flled grey histogram) was determined in comparison to the negative control staining (black lined histogram) served as a control. Te purity of isolated immune cells (Becton Dickinson, San Jose, CA, USA). Data analysis was determined using the antibodies CD3 FITC, CD4 was performed using FlowJo software (TreeStar Inc., PE, CD8 PE, CD14 PE and CD15 FITC. CCR9 expression Ashland, OR, USA). Gating strategies for PBMCs and was investigated separately on all cell types using an anti- macrophages are shown in Fig. 6. body against CCR9 (CD199 APC). Te PBMCs contained 60% T-lymphocytes ­(CD3+), 46% ­CD4+ T-lymphocytes, Cytokine detection 26% ­CD8+ T-lymphocytes and 19% monocytes ­(CD14+). A LEGENDplex™ human infammation panel (13-plex) Additionally, the PBMCs exhibited a minor contamina- assay (BioLegend, San Diego, USA) was performed tion with CD15­ + granulocytes (2%). Te purifed CD4­ + according to the manufacturer´s instructions to investi- T-lymphocytes were shown to be 86% ­CD4+ with small gate the secretion of diferent cytokines (IFN-γ, TNF-α, amounts of CD8­ + (7%), CD14­ + (4%) and CD15­ + (2%) MCP-1, IL-6, IL-8, IL-10) from the cultivated and polar- cells. Te purity of the isolated CD8­ + T-lymphocytes ized macrophages and PBMCs after stimulation. Briefy, was found to be 99%. Te surface marker changes on lyophilized human infammation panel standard cock- macrophages were analyzed with the antibodies CD14 tail was reconstituted with 250 μl assay bufer for 10 min APCCy7 and CD16 PerCPCy5.5 (general monocyte/ and a standard series from 2.4 pg/ml to 10,000 pg/ml macrophage markers), CD80 PE and HLA-DR PECy7 was prepared. Te pre-mixed beads bottle was vortexed (M1 macrophage polarization markers), and CD206 APC for 2 min before use. 25 μl of the following reagents were (M2 polarization marker). PBMCs were also stained with pipetted into each microtube: assay bufer, standard or HLA-DR PECy7. Additionally, the LIVE/DEAD™ Fix- samples, mixed beads, and detection antibodies. All sam- able Aqua Dead Cell Stain Kit (Termo Fisher Scientifc, ples were measured on a FACS Canto II fow cytometer Waltham, USA) was used to exclude dead cells. All anti- and analyzed using the LEGENDplex™ software (BioLeg- bodies were purchased from BioLegend, Miltenyi Biotec end, San Diego, USA). and Becton Dickinson; dilutions were prepared accord- ingly to the manufacturers recommendations. After Chemotaxis assay antibody incubation, the samples were washed with cold Migration was investigated using a modifed Boyden- FACS bufer, resuspended in 1% paraformaldehyde (PFA; Chamber migration assay. 96-multiwell format Chem- Roth, Karlsruhe, Germany) in FACS bufer and trans- oTx plates with polycarbonate membranes (MERCK ferred to 5 ml FACS tubes (Falcon). Samples were kept Millipore, Burlington, USA) were used. Te pore sizes at 4 °C in the dark until measurement. Data was acquired were chosen for each cell type individually to enable using a FACS Canto II device with FACS Diva software active migration without cells passing through the pores Spinnen et al. J Nanobiotechnol (2021) 19:83 Page 15 of 16

passively. Diameters of 3 μm were chosen for T-cells, views and opinions expressed within this manuscript are those of all authors. All authors read and approved the fnal manuscript. 5 μm for monocytes and PBMCs, and 8 μm for mac- rophages. Except for the macrophages, which were culti- Funding vated, polarized and detached as described previously, all Open Access funding enabled and organized by Projekt DEAL. This research project was funded by the BIH Centre for Regenerative Therapies (Funding of cell types were freshly isolated. A CCL25 dilution series Federal Ministry of Education and Research; BMBF Grant #13GW0099). (0.01–1000 nM) in complete cell culture medium with- out M-CSF was prepared on ice to enable comparison of Availability of data and materials The datasets used and analyzed during the current study are available from the chemotactic efect between particles, supernatants the corresponding author on reasonable request. In general, data generated and pure CCL25. Te bufer of the tested release samples or analyzed during this study are included in this published article and its was exchanged as described above. To address the pos- additional information fles. sibility of an inhibitory efect of the release samples on the migration of the immune cell types, the samples were Declarations tested undiluted and in 1:10, 1:100 and 1:1000 dilutions. Ethics approval and consent to participate Since a chemotactic reaction of the immune cells could Study was approved by ethics committee of Charité–Universitätsmedizin Ber- also be attributed to the PLGA fragments in the samples, lin, (Internal Review Number EA2/139/10; EA1/226/14) after informed consent. NL release samples from the same days were tested as a Consent for publication comparison. To quantify the number of migrated cells, an Not applicable. image of each well was taken with the software ProgRes ™ Competing interests Capture Pro (Jenoptik, Jena, Germany). Te cell num- Michael Sittinger, Kristin Fröhlich and Jochen Ringe are inventors of the follow- bers in each picture were determined using the ImageJ ing patents: US 9533297B2; EP 2645998B1. plug-in Cell ImageAnalyzer v4.5. Te program subtracted Author details the red value from the green value in the RGB color 1 Tissue Engineering Laboratory, BIH Center for Regenerative Therapies, mode, yielding a grey-scale image. Te particle analyzer Department for Rheumatology and Clinical Immunology & Berlin Institute counted the cells using diferent thresholds for the detec- of Health at Charité–Universitätsmedizin Berli, BCRT​, Charitéplatz 1, 10117 Ber- lin, Germany. 2 Institute of Medical Immunology and Berlin Institute of Health tion, starting from the adjusted ‘Max-Entropy Treshold’ Center for Regenerative Therapies, Institute of Medical Immunology, Charité– in ImageJ. Universitaetsmedizin Berlin, corporate member of Freie Universitaet Berlin and Humboldt-Universitaet Zu Berlin, Augustenburger Platz 1, 13353 Berlin, Germany. 3 DZHK (German Center for Cardiovascular Research), partner site Statistical analysis Berlin, Germany. Microsoft Excel (2011) was used to test datasets for nor- Received: 7 January 2021 Accepted: 10 March 2021 mal distribution using the Lillefors-corrected Kolmogo- row-Smirnov-Test. Normally distributed datasets were analyzed using a paired student’s t-test to detect signif- cant changes of receptor expression, cytokine secretion References and cell migration of CCL25 stimulated immune cell 1. Aigner T, et al. Suppression of cartilage matrix gene expression in upper subsets compared to negative controls. Datasets which zone chondrocytes of osteoarthritic cartilage. Arthritis Rheum Of J Am Coll Rheumatol. 1997;40:562–9. were not normally distributed were tested for signif- 2. Rezuș E, et al. 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